Protease deletion

ABSTRACT

A Bacillus cell contains a mutation in the epr gene resulting in inhibition of the production by the cell of the proteolytically active epr gene product; the cell may further contain mutations in the genes encoding proteolytically active residual protease I (RP-I) and proteolytically active residual protease II (RP-II) (mpr).

This is a divisional of application Ser. No. 07/396,521, filed Aug. 21,1989, which is a continuation-in-part of application Ser. No.07/347,428, filed May 4, 1989, which is a continuation-in-part ofapplication Ser. No. 07/273,423, filed Nov. 18, 1988, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to Bacillus strains useful for the expression andsecretion of desired polypeptides (as used herein, "polypeptide" meansany useful chain of amino acids, including proteins).

Bacillus strains have been used as hosts to express heterologouspolypeptides from genetically engineered vectors. The use of a Grampositive host such as Bacillus avoids some of the problems associatedwith expressing heterologous genes in Gram negative organisms such as E.coli. For example, Gram negative organisms produce endotoxins which maybe difficult to separate from a desired product. Furthermore, Gramnegative organisms such as E. coli are not easily adapted for thesecretion of foreign products, and the recovery of products sequesteredwithin the cells is time-consuming, tedious, and potentiallyproblematic. In addition, Bacillus strains are non-pathogenic and arecapable of secreting proteins by well-characterized mechanisms.

A general problem in using Bacillus host strains in expression systemsis that they produce large amounts of proteases which can degradeheterologous polypeptides before they can be recovered from the culturemedia. The proteases which are responsible for the majority of thisproteolytic activity are produced at the end of the exponential phase ofgrowth, under conditions of nutrient deprivation, as the cells preparefor sporulation. The two major extracellular proteases an alkalineserine protease (subtilisin), the product of the apr gene, and a neutralmetalloprotease, the product of the npr gene, are secreted into themedium, whereas the major intracellular serine protease, Isp-1, isproduced within the cells. Other investigators have created geneticallyaltered Bacillus strains that produce below-normal levels of one or moreof these three proteases, but these strains still produce high enoughlevels of protease to cause the degradation of heterologous geneproducts prior to purification.

Stahl et al. (J. Bact., 1984, 158:411) disclose a Bacillus proteasemutant in which the chromosomal subtilisin structural gene was replacedwith an in vitro derived deletion mutation. Strains carrying thismutation produced only 10% of the wild-type extracellular serineprotease activity. Yang et al. (J. Bact., 1984, 160:15) disclose aBacillus protease mutant in which the chromosomal neutral protease genewas replaced with a gene having an in vitro derived deletion mutation.Fahnestock et al. (WO 86/01825) describe Bacillus strains lackingsubtilisin activity which were constructed by replacing the nativechromosomal gene sequence with a partially homologous DNA sequencehaving an inactivating segment inserted into it. Kawamura et al. (J.Bact., 1984, 160:442) disclose Bacillus strains carrying lesions in thenpr and apr genes and expressing less than 4% of the wild-type level ofextracellular protease activity. Koide et al. (J. Bact., 1986, 167:110)disclose the cloning and sequencing of the isp-1 gene and theconstruction of an Isp-1 negative mutant by chromosomal integration ofan artificially deleted gene.

Genetically altered strains which are deleted for the extracellularprotease genes (apr and npr) produce significantly lower levels ofprotease activity than do wild-type Bacillus strains. These bacteria,when grown on medium containing a protease substrate, exhibit little orno proteolytic activity, as measured by the lack of appearance of a zoneof clearing (halo) around the colonies. Some hetetologous polypeptidesand proteins produced from these double mutants are, nevertheless,substantially degraded prior to purification, although they are morestable than when produced in a wild-type strain of Bacillus.

SUMMARY OF THE INVENTION

The invention provides improved Bacillus cells containing mutations inone or more of three previously uncharacterized protease genes; thecells also preferably contain mutations in the apr and npr genes thatencode the major extracellular proteases, resulting in the inhibition bythe cells of production of these extracellular proteases. The mutationsof the invention include a mutation in the epr gene which inhibits theproduction by the cell of the proteolytically active epr gene product, amutation in the gene (herein, the "RP-I" gene) encoding residualprotease I (RP-I) which inhibits the production by the cell ofproteolytically active RP-I, and a mutation in the gene (herein, the"RP-II" gene mpr) encoding residual protease II (RP-II). The proteasesencoded by the epr gene and RP-II genes are novel proteins. Mostpreferably, the mutations of the invention are deletions within thecoding region of the genes, including deletion of the entire codingregion; alternatively, a mutation can consist of a substitution of oneor more base pairs for naturally occuring base pairs, or an insertionwithin the protease coding region.

The Bacillus cells of the invention may also contain a mutation in theisp-1 gene encoding intracellular serine protease I and may in additioncontain a mutation which blocks sporulation and thus reduces the cell'scapacity to produce sporulation-dependent proteases; preferably, thismutation blocks sporulation at an early stage but does not eliminate thecell's ability to be transformed by purified DNA; most preferably, thismutation is the spoOA mutation (described below). The invention furtherprovides a method for producing stable heterologous polypeptides in aBacillus host cell by modifying the host to contain mutations in the aprand npr genes and in one or more of the genes including the epr gene,the RP-I gene, and the RP-II gene.

The invention also features purified DNA, expression vectors containingDNA, and host Bacillus cells transformed with DNA encoding any of theproteases RP-I, RP-II, or the product of the epr gene; preferably, suchDNA is derived from Bacillus subtilis.

The invention also features the isolation of substantially pure Epr,residual protease I (RP-I), and another previously uncharacterizedprotease called residual protease II (RP-II), and characterization ofthe RP-I and RP-II proteases; as used herein, "substantially pure" meansgreater than 90% pure by weight.

The terms "epr gene", "RP-I gene", and "RP-II gene" herein mean therespective genes corresponding to these designations in Bacillussubtilis, and the evolutionary homologues of those genes in otherBacillus species, which homologues, as is the case for other Bacillusproteins, can be expected to vary in minor respects from species tospecies. The RP-I and RP-II genes of B. subtilis are also designated,respectively, the bpr and mpr genes. In many cases, sequence homologybetween evolutionary homologues is great enough so that a gene derivedfrom one species can be used as a hybridization probe to obtain theevolutionary homologue from another species, using standard techniques.In addition, of course, those terms also include genes in which basechanges have been made which, because of the redundancy of the geneticcode, do not change the encoded amino acid residue.

Using the procedures described herein, we have produced Bacillus strainswhich are significantly reduced in their ability to produce proteases,and are therefore useful as hosts for the expression, withoutsignificant degradation, of heterologous polypeptides capable of beingsecreted into the culture medium. We have found that the Bacillus cellsof the invention, even though containing several mutations in genesencoding related activities, are not only viable but healthy.

Any desired polypeptide can be expressed according to the invention,e.g., medically useful proteins such as hormones, vaccines, antiviralproteins, antitumor proteins, antibodies or clotting proteins; andagriculturally and industrially useful proteins such as enzymes orpesticides, and any other polypeptide that is unstable in Bacillus hoststhat contain one or more of the proteases inhibited according to thepresent invention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF PREFERRED EMBODIMENTS

The drawings will first be briefly described.

DRAWINGS

FIG. 1 is a series of diagrammatic representations of the plasmids p371and p371Δ, which contain a 2.4 kb HindIII insert encoding the Bacillussubtilis neutral protease gene and the same insert with a deletion inthe neutral protease gene, respectively, and p371ΔCM, which contains theBacillus cat gene.

FIG. 2 is a Southern blot of HindIII digested IS75 and IS75NΔDNA probedwith a ³² P-labeled oligonucleotide corresponding to part of thenucleotide sequence of the npr gene.

FIG. 3 is a representation of the 6.5 kb insert of plasmid pAS007, whichencodes the Bacillus subtilis subtilisin gene, and the construction ofthe deletion plasmid pAS13.

FIG. 4 is a representation of the plasmid pISP-1 containing a 2.7 kbBamHI insert which encodes the intracellular serine protease ISP-1, andthe construction of the ISP-1 deletion plasmid pAL6.

FIG. 5 is a diagrammatic representation of the cloned epr gene, showingrestriction enzyme recognition sites.

FIG. 6 is the DNA sequence of the epr gene.

FIG. 7 is a diagrammatic representation of the construction of theplasmid pNP9, which contains the deleted epr gene and the Bacillus catgene.

FIG. 8 is the amino acid sequence of the first 28 residues of RP-I andthe corresponding DNA sequence of the probe used to clone the RP-I gene.

FIG. 9 is a restriction map of the 6.5 kb insert of plasmid pCR83, whichencodes the RP-I protein.

FIGS. 10a and 10b are the DNA sequence of DNA encoding RP-I protease.

FIG. 11 is the amino acid sequence of three internal RP-II fragments (a,b, c), and the nucleotide sequence of three guess-mers used to clone thegene (a), (b) and (c).

FIG. 12A-B are a Southern blot of GP241 chromosomal DNA probed withBRT90 and 707.

FIG. 13 is a diagram of (a) a restriction map of the 3.6 kb PstI insertof pLPI, (b) the construction of the deleted RP-II gene and (c) theplasmid used to create an RP-II deletion in the Bacillus chromosome.

FIG. 14 is the DNA sequence of DNA encoding RP-II.

GENERAL STRATEGY FOR CREATING PROTEASE DELETED BACILLUS STRAINS

The general strategy we followed for creating a Bacillus strain which issubstantially devoid of proteolytic activity is outlined below.

A deletion mutant of the two known major extracellular protease genes,apr and npr, was constructed first. The isp-1 gene encoding the majorintracellular protease was then deleted to create a triple proteasedeletion mutant. The spoOA mutation was introduced into either thedouble or triple deletion mutants to significantly reduce anysporulation dependent protease activity present in the cell. A geneencoding a previously unknown protease was then isolated and its entirenucleotide sequence was determined. The gene, epr, encodes a primaryproduct of 645 amino acids that is partially homologous to bothsubtilisin (Apr) and the major internal serine protease (Isp-1) of B.subtilis. A deletion of this gene was created in vitro and introducedinto the triple protease deleted host. A deletion in a newly identifiedgene encoding residual protease RP-I was then introduced to create astrain of B. subtilis having substantially reduced protease activity andexpressing only the RP-II activity. RP-II has been purified and aportion of the amino acid sequence was determined for use in creatingthe nucleic acid probes which were used to clone the gene encoding thisprotease. Upon cloning the gene, it was possible to create a Bacillusstrain which contains a deletion in the RP-II gene and is thus incapableof producing RP-II.

Detailed procedures for construction of the protease gene deletions andpreparation of Bacillus strains exhibiting reduced protease activity aredescribed below.

General Methods

Our methods for the construction of a multiply deleted Bacillus strainare described below. Isolation of B. subtilis chromosomal DNA was asdescribed by Dubnau et al., (1971, J. Mol. Biol., 56:209). B. subtilisstrains were grown on tryptose blood agar base (Difco Laboratories) orminimal glucose medium and were made competent by the procedure ofAnagnostopoulos et al., (J. Bact., 1961, 81:741 ). E. coli JM107 wasgrown and made competent by the procedure of Hanahan (J. Mol. Biol.,1983, 166:587). Plasmid DNA from B. subtilis and E. coli were preparedby the lysis method of Birnboim et al. (Nucl. Acid. Res., 1979, 7:1513).Plasmid DNA transformation in B. subtilis was performed as described byGryczan et al., (J. Bact., 1978, 134: 138).

Protease Assays

Two different protease substrates, azocoll and casein (Labelled eitherwith ¹⁴ C or the chromophore resorufin), were used for protease assays,with the casein substrate being more sensitive to proteolytic activity.Culture supernatant samples were assayed either 2 or 20 hours intostationary phase. Azocoll-based protease assays were performed by adding100 ul of culture supernatant to 900 ul of 50 mM Tris, pH 8, 5 mM CaCl₂,and 10 mg of azocoll (Sigma), a covalently modified, insoluble form ofthe protein collagen which releases a soluble chromophore whenproteolytically cleaved. The solutions were incubated at 37° C. for 30minutes with constant shaking. The reactions were then centrifuged toremove the insoluble azocoll and the A₅₂₀ of the solution determined.Inhibitors were pre-incubated with the reaction mix for 5 minutes at 37°C. Where a very small amount of residual protease activity was to bemeasured, ¹⁴ C-casein or resorufin-labelled casein was used as thesubstrate. In the ¹⁴ C-casein test, culture supernatant (100 ul) wasadded to 100 ul of 50 mM Tris, 5 mM CaCl₂ containing 1×10⁵ cpm of ¹⁴C-casein (New England Nuclear). The solutions were incubated at 37° C.for 30 minutes. The reactions were then placed on ice and 20 ug of BSAwere added as carrier protein. Cold 10% TCA (600 ul) was added and themix was kept on ice for 10 minutes. The solutions were centrifuged tospin out the precipitated protein and the supernatants counted in ascintillation counter. The resorufin-labelled casein assay involvedincubation of culture supernatant with an equal volume ofresorufin-labelled casein in Tris=Cl buffer, pH 8.0, at 37° C. forvarious times. Following incubation, unhydrolyzed substrate wasprecipitated with TCA and the resulting chromogenic supernatant wasquantitated spectrophotometrically.

Deletion of the npr Gene

According to Yang et al. (J. Bact., 1984, 160: 15), the npr gene iscontained within overlapping EcoRI and HindIII restriction fragments ofB. subtilis DNA, and a majority of the gene sequence is located on the2.4 kb HindIII fragment. This fragment was chosen for creation of thenpr deletion.

An individual clone containing the 2.4 kb HindIII fragment was isolatedfrom a clone bank of genomic HindIII fragments prepared as follows.Chromosomal DNA was isolated from B. subtilis strain IS75, digested withHindIII and size fractionated by electrophoresis on a 0.8% agarose gel.DNA in the 2-4 kb size range was electroeluted from the gel. Thepurified DNA was ligated to HindIII digested and alkaline phosphatasetreated pUC9 DNA (an E. coli replicon commercially available fromBethesda Research Labs, Rockville, Md.), transformed into competentcells of E. coli strain JM107, and plated on LB+50 ug/ml ampicillinresulting in 1000 Amp^(R) colonies.

Colonies containing the cloned neutral protease gene fragment wereidentified by standard colony hybridization methods (Maniatis et al.,1983, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor,N.Y.). Briefly, transformants are transferred to nitrocellulose filters,lysed to release the nucleic acids and probed with an nor specificprobe. A 20 base oligonucleotide complementary to the npr gene sequencebetween nucleotides 520 and 540 (Yang et al., supra) was used as theprobe. The sequence is 5'GGCACGCTTGTCTCAAGCAC 3'. A representative clonecontaining the 2.4 kb HindIII insert was identified and named p371 (FIG.1).

A deleted form of the npr gene in p371 was derived in vitro. A 580 bpinternal RsaI fragment was deleted by digesting p371 DNA with RsaI andHindIII. The 600 bp HindIII-RsaI fragment spanning the 5' end of thegene and the 1220 bp RsaI-HindIII fragment spanning the 3' end of thegene (see FIG. 1) were isolated and cloned into HindIII and alkalinephosphatase treated pUC9. This resulted in the deletion of the centerportion of the npr gene. The ligated DNA was transformed into E. coliJM107. A clone having the desired deletion within the npr gene wasidentified by restriction enzyme analysis. This plasmid is designatedp371Δ.

A gene encoding a selectable marker was included on the vector tofacilitate the selection of integrants in Bacillus. The Bacillus catgene, encoding resistance to chloramphenicol (Cm^(r)), was isolated fromplasmid pMI1101 (Youngman et al., 1984, Plasmid 12:1-9) on a 1.3 kb SalIfragment and cloned into the SalI site of p371Δ. This DNA wastransformed into E. coli JM107 and transformants were screened forchloramphenicol resistance. A representative plasmid containing both thedeleted npr gene and the cat gene was named p371ΔCm (FIG. 1).

The vector p371ΔCm was derived from the E. coli replicon pUC19 and istherefore unable to replicate in a Bacillus host. The wild-type npr genein the chromosome of the recipient host was exchanged for the deletednpr gene contained on the vector by reciprocal recombination betweenhomologous sequences. The Cm^(r) marker gene enabled the selection ofcells into which the vector, inclusive of the protease gene sequence,had integrated.

Vector sequences that integrated with the deleted npr gene werespontaneously resolved from the chromosome at a low frequency, taking acopy of the npr gene along with them. Retention of the deleted proteasegene in the chromosome was then confirmed by assaying for the lack ofprotease activity in the Cm^(s) segregants.

Specifically, competent B. subtilis IS75 cells were transformed withp371ΔCm and selected for Cm^(r). Approximately 2000 colonies, which hadpresumably integrated the deleted npr gene adjacent to, or in place of,the wild type gene, were selected which were resistant tochloramphenicol. Approximately 25% of the colonies formed smaller zonesof clearing on starch agar indicating that the wild-type gene had beenreplaced with the deleted form of the gene. No neutral protease activitywas detected in supernatants from these cell cultures. In contrast, highlevels of neutral protease activity were assayed in culture fluids fromwild type IS75 cells. Segregants which contained a single integratedcopy of the deleted protease gene, but which had eliminated the vectorsequences were then selected as follows.

A culture of Cm^(r) colonies was grown overnight in liquid media withoutselection then plated onto TBAB media. These colonies were thenreplicated onto media containing chloramphenicol and those that did notgrow in the presence of chloramphenicol were identified and selectedfrom the original plate. One such Npr negative colony was selected anddesignated IS75NΔ.

Deletion within the npr gene in IS75NΔ was confirmed by standardSouthern blot analysis (Southern, 1977, J. Mol. Biol. 98:503) of HindIIIdigested DNA isolated from B. subtilis IS75N and IS75NΔ probed with the³² P-labelled npr-specific oligonucleotide. The probe hybridized with a2.4 kb HindIII fragment in wild-type IS75N DNA and with a 1.8 kbfragment in IS75N Δ DNA indicating that 600 bp of the npr gene weredeleted in IS75NΔ (see FIG. 2).

Deletion of the apr Gene

To clone the subtilisin gene (apr) a genomic library from IS75 DNA wasfirst prepared. Chromosomal DNA was isolated and digested with EcoRI andseparated by electrophoresis through a 0.8% agarose gel. Fragments inthe 5-8 kb size range were purified by electroelution from the gel. Thefragments were ligated with EcoRI digested pBR328 DNA (publiclyavailable from New England BioLabs) and transformed into competent E.coli JM107 cells. Transformants were screened for plasmids containingapr gene inserts by hybridizing with a synthetic ³² P-labelled 17-meroligonucleotide probe which was complementary to the apr gene sequencebetween nucleotides 503 and 520 (Stahl et al., 1984, J. Bact. 158:411).A clone with a 6.5 kb EcoRI insert that hybridized with the probe wasselected and named pAS007 (FIG. 3). The 6.5 kb fragment contained theentire coding sequence of the subtilisin gene.

A mutant of the apr gene was created by deleting the two internal HpaIfragments (FIG. 3). pAS007 was first digested with HpaI and thenrecircularized by ligating in a dilute solution (5 ug/ml) to eliminatethe two HpaI fragments. Approximately 200 Amp^(r) colonies arosefollowing transformation of JM107 cells. One of these transformantscontained a 4.8 kb EcoRI insert with one internal HpaI site. It wasdesignated pAS12. The deletion in the apr gene extended 500 bp beyondthe 3' end of the gene, however this DNA apparently did not contain anygenes that were essential to B. subtilis.

A 1.3 kb SalI fragment containing the Bacillus cat gene was cloned intothe SalI site of pAS12 (described above) for selection of integrants inthe Bacillus host chromosome. The plasmid DNA was transformed into E.coli JM107, plated on media containing ampicillin and approximately 50Amp^(r) colonies were recovered and replica plated onto media containing7.5 ug/ml chloramphenicol. Three of the 50 colonies were Cm^(r) .Plasmid DNA was isolated from these three clones and analyzed byrestriction digestion. One of the plasmids had the desired restrictionpattern and was named pAS13 (FIG. 3).

To promote integration of the deleted protease gene into the B. subtilischromosome, pAS13 was introduced into strain IS75NΔ and selected forCm^(r) transformants. The transformants were then screened forreplacement of the wild-type apr gene with the deleted gene by platingon TBAB plates containing 5 ug/ml Cm and 1.5% casein. Several of thecolonies which did not produce halos were selected for loss of theCm^(r) gene as described above. A representative transformant was chosenand designated GP199.

Protease activity was assayed in the culture fluids from the doubleprotease deleted strain, as well as in the strain having only thedeleted neutral protease gene. Protease activity in Npr⁻, Apr⁻ mutantcells was approximately 4-7% of wild type levels whereas the Npr⁻ mutantexhibited higher levels of protease activity.

amyE Mutation

Protease deficient strains were tested in connection with the productionof a Bacillus amylase. To assay the levels of amylase produced byvarious plasmid constructs it was necessary to introduce a mutantamylase gene into the host in place of the wild type gene. This step isnot essential to the present invention and does not affect the level ofprotease activity; it was performed only because plasmid encoded amylaselevels could not be determined in the presence of the chromosomallyencoded amylase. The amyE allele was transformed from B. subtilis strainJF206 (trpC2, amyE) into GP199 by a transformation/selection processknown as congression. This process relies on the ability of competent B.subtilis cells to be transformed by more than one piece of chromosomalDNA when the transforming DNA is provided in excess. The processinvolves initial selection of competent cells in the population byassaying for expression of a selectable marker gene which subsequentlyfacilitates screening for co-transfer of an unselectable marker, such asinability to produce amylase.

Total chromosomal DNA was isolated from JF206 or a similar straincontaining an amy E mutation. Saturating concentrations (˜lug) weretransformed into competent GP199 (met⁻, leu⁻, his⁻) and His⁺transformants were selected on minimal media supplemented withmethionine and leucine. The transformants were screened for an amylaseminus phenotype on plates having a layer of top agar containingstarch-azure. Five percent of the His⁺ colonies were unable to producehalos indicating that the amylase gene was defective. One suchtransformant was assayed for the protease-deficient phenotype and wasdesignated GP200.

Supernatant samples from cultures of the double protease mutant wereassayed for protease activity using azocoll as the substrate. Whenassayed on this substrate, protease activity in the double proteasemutant strain was 4% of wild type levels. When the more sensitivesubstrate ¹⁴ C-casein was used in the protease assay, the double mutantdisplayed 5-7% of the wild type B. subtilis activity. Although proteaseactivity in this strain was low, we discovered that certain heterologousgene products produced by these protease deficient cells were notstable, indicating the presence of residual protease activity. We thensought to identify and mutate the gene(s) responsible for the residualprotease activity.

In order to characterize the residual protease activity, a number ofknown protease inhibitors were tested for their ability to reduceprotease levels in cultures of the double protease mutant strain. PMSF(phenylmethylsulfonyl flouride), a known inhibitor of serine proteaseactivity, was found to be the most effective. The addition of PMSF togrowing cultures of Apr⁻ Npr⁻ Bacillus cells successfully increased thestability of heterologous peptides and proteins synthesized in andsecreted from these cells. These results indicated that at least aportion of the residual degradative activity was due to a serineprotease.

Subtilisin is the major serine protease to be secreted by B. subtilis;however, the serine protease encoded by the isp-1 gene (ISP-1) has beenshown to accumulate intracellularly during sporulation (Srivastava etal., 1981, Arch. Microbiol., 129:227). In order to find out if theresidual protease activity was due to Isp-1, a deleted version of theisp-1 gene was created in vitro and incorporated into thedouble-protease deleted strain.

Deletion of the isp-1 Gene

The isp-1 gene is contained within a 2.7 kb BamHI fragment of B.subtilis chromosomal DNA (Koide et al., 1986, J. Bact., 167:110).Purified DNA was digested with BamHI and fragments in the 2.7 kb sizerange were electroeluted from an agarose gel, ligated into BamHIdigested pBR328 and transformed into E. coli JM107 cells. One Amp^(r)colony that produced a halo on LB media containing 1% casein wasselected and named pISP-1. Restriction analysis of the DNA indicatedthat pISP-1 carried a 2.7 kb BamHI insert which hybridized with asynthetic 25 base ³² P-labeled oligonucleotide probe5'ATGAATGGTGAAATCCGCTTGATCC 3'! complementary to the isp-1 gene sequence(Koide et al, supra). The restriction pattern generated by SalI andEcoRI digestions confirmed the presence of the isp-1 gene in pISP-1.

A deletion was created within the isp-1 gene by taking advantage of aunique SalI site located in the center of the gene. Because there was anadditional SalI site in the vector, the 2.7 kb BamHI gene insert wasfirst cloned into the BamHI site of a derivative of pBR322 (pAL4) fromwhich the SalI site had been eliminated (FIG. 4). The resulting plasmid,pAL5, therefore had a unique SalI site within the isp-1 gene. pAL5 DNAwas digested with SalI, treated with Bal31 exonuclease for five minutesat 37° C. to delete a portion of the gene sequence, and religated. TheDNA was transformed into JM107 and resulting Amp^(r) colonies werescreened for a BamHI insert of reduced size. A plasmid with a 1.2 kbdeletion within the BamHI insert was selected and named pAL6 (FIG. 4).

The cat gene was purified from the E. coli plasmid pMI1101 on a SalIfragment as above and cloned into pAL6 at the EcoRV site. The resultingDNA was transformed into the double protease mutant strain (GP200) andintegrants containing the deleted ISP-1 gene were selected as describedabove. The triple-protease deleted strain is called GP208 (aprΔ, nprΔ,isp-1Δ). Using a casein substrate, protease activity was measured in thetriple-mutant strain (Apr⁻, Npr⁻, Isp-1⁻) and found to be 4% of the wildtype level, about the same as the double mutant strain.

The remaining 4% residual protease activity was apparently due either toa' previously described esterase called bacillopeptidase F (Roitsch etal., 1983, J. Bact., 155:145), or to previously unknown and unidentifiedprotease gene(s).

Introduction of a Sporulation Mutation

Because it had been shown that the production of certain proteases wasassociated with the process of sporulation in B. subtilis, we reasonedthat it may be useful to include a mutation which blocked sporulation inour protease deficient hosts and thus further reducesporulation-dependent protease production in these strains. Mutationsthat block the sporulation process at stage 0 reduce the level ofprotease produced, but do not eliminate the ability of the cells to betransformed by purified DNA. spoOA mutations have been shown to beparticularly efficient at decreasing protease synthesis (Ferrari et al.,1986, J. Bact. 166:173).

We first introduced the spoOA mutation into the double proteasedeficient strain as one aspect of our strategy to eliminate theproduction of the serine protease, Isp-1 . We ultimately introduced thespoOA mutation into the triple- and quadruple- protease deficientstrains. This feature of our invention is useful only when a promoter,contained within an expression vector for the production of heterologousgene products in a Bacillus host, is not a sporulation-specific promoter(e.g. the spoVG promoter).

Saturating amounts of chromosomal DNA were prepared from B. subtilisstrain JH646 (spoOA, Prot⁺, Amy⁺, Met⁺) or similar strains having aspoOA mutation, and transformed into competent GP200 cells (Spo⁻, Prot⁻,Amy⁻, Met⁻). Met⁺ transformants were selected by growth on minimal mediaplates. Resulting transformants were then screened for co-transformationof the spoOA allele by assaying on sporulation medium (Difco) for thesporulation deficiency phenotype, characterised by smooth colonymorphology and the lack of production of a brown pigment. Approximately9% of the Met⁺ transformants appeared to be co-transformed with thespoOA allele; a number of these were rescreened on plates containingeither starch-azure or casein to confirm that the recipients had notalso been co-transformed with intact amylase or protease genes from thedonor DNA. One transformant that did not exhibit detectable proteaseactivity was designated GP205 (spoOA, amyE, aprA, nprE). Protease levelsproduced by this host were 0.1% of the level found in the extracellularfluid of the Spo⁺ host, when casein was the substrate.

In the same manner, the spoOA mutation was introduced into the tripleprotease deficient mutant GP208 (aprΔ, nprΔ, isp-1Δ) and the quadrupleprotease deficient mutant GP216 (aprΔ, nprΔ, isp-1Δ, eprΔ and describedbelow). The resulting Spo⁻ strains are GP210 and GP235, respectively.These strains are useful when the expression vector is not based on asporulation dependent promoter.

Identification of a New Protease Gene

We expected that the isolation and cloning of the gene(s) responsiblefor the remaining protease activity would be difficult usingconventional methods because cells did not produce large enough amountsof the enzyme(s) to detect by the appearance of halos on casein plates.We reasoned that it should be possible to isolate the gene(s) if it werereplicated on a high-copy vector so that the copy number of the gene(s),and thus protease production, would be amplified to detectable levels.This strategy enabled us to isolate a novel protease gene from aBacillus gene bank. The first of these new protease genes has been namedepr (extracellular protease). Deletion mutants of this new gene werederived in vitro and introduced into the Apr⁻ Npr⁻ Isp⁻ Bacillus hoststrains by gene replacement methods as described above.

Cloning the epr Gene

In order to obtain a clone carrying a gene responsible for residualprotease activity, a Sau3A library of B. subtilis GP208 DNA wasprepared. Chromosomal DNA was isolated, subjected to partial digestionwith Sau3A and size-fractionated on an agarose gel. Fragments in the 3-7kb size range were eluted from the gel and cloned into the BglII site ofpEc224, a shuttle vector capable of replicating in both E. coli andBacillus (derived by ligating the large EcoRI-PvuII fragment of pBR322with the large EcoRI-PvuII fragment of pBD64 (Gryczan et al., 1978, PNAS75:1428)). The ligated DNA was transformed into E. coli JM107 and platedon media containing casein. None of the 1200 E. coli colonies producedhalos on casein plates, however by restriction analysis of the purifiedplasmid DNA, approximately 90% of the clones contained inserts with anaverage size of about 4 kb. The clones were transformed into a Bacillushost to screen for protease activity as follows. E. coli transformantswere pooled in twelve groups of 100 colonies each (G1-G12). The pooledcolonies were grown in liquid media (LB+50 ug/ml ampicillin), plasmidDNA was isolated, transformed into B. subtilis. GP208 (aprΔ, nprΔ,isp-1Δ) and plated on casein plates. Halos were observed aroundapproximately 5% of transformants from pool G11. Plasmid DNA wasisolated from each of the positive colonies and mapped by restrictionenzyme digestion. All of the transformants contained an identical insertof approximately 4 kb (FIG. 5). One of these plasmids was selected andnamed pNP1.

Characterization of epr Protease Activity

The residual protease activity remaining in GP208 (aprΔ, nprΔ, isp-1Δ)cultures accounted for only a small percentage of the total proteaseactivity produced by the host. In order to characterize the type ofprotease encoded by the epr gene, the effect of different inhibitors onthe protease secreted by B. subtilis GP208/pNP1 was examined.

Culture media was obtained two hours into stationary phase and assayedusing ¹⁴ C-casein as the substrate. The level of protease activitypresent in GP208 was not high enough to detect in the standard proteaseassay described above, however, appreciable protease activity wasdetected in the culture medium of GP208/pNP1, carrying the amplified eprgene. The epr protease activity was inhibited in the presence of both 10mM EDTA and 1 mM PMSF suggesting that it encodes a serine protease whichrequires the presence of a cation for activity. (Isp-1, another serineprotease, is also inhibited by EDTA and PMSF.)

Subcloning the epr Gene

A 2.7 kb HpaI-SalI subfragment was isolated from the pNP1 insert andcloned into pBs81/6, a derivative of pBD64 (derived by changing thePvuII site to a HindIII site using synthetic linkers). Transformantscarrying this subcloned fragment were capable of producing halos oncasein plates, indicating that the entire protease gene was presentwithin this fragment. A representative clone was named pNP3.

The location of the gene within the pNP3 insert was further defined bysubcloning a 1.6 kb EcoRV subfragment into pBs81/6 and selecting for thecolonies producing halos on casein plates. A clone which produced ahalo, and which also contained the 1.6 kb insert shown in FIG. 5, wasdesignated pNP5. The presence of the protease gene within this fragmentwas confirmed by deleting this portion of the 4 kb insert from pNP1.pNP1 was digested with EcoRV and religated under conditions whichfavored recircularization of the vector without incorporation of the 1.6kb EcoRV insert. The DNA was transformed into GP208 and colonies werescreened on casein plates. Greater than 95% of the transformants did notproduce halos, indicating that the protease gene had been deleted fromthese clones. A representative clone was selected and is designatedpNP6. (The small percentage of colonies that produced halos werepresumed to have vectors carrying the native epr gene resulting fromrecombination between the chromosomal copy of the gene and homologoussequences within the plasmid.)

Nucleotide and Deduced Amino Acid Sequence of the epr Gene

Subcloning and deletion experiments established that most of theprotease gene was contained on the 1.6 kb EcoRV fragment (FIG. 5).Determination of the nucleotide sequence of the 1.6 kb EcoRV fragment(FIG. 6) revealed an open reading frame which covered almost the entirefragment starting 450 bp from the left end and proceeding through theright end (see FIG. 2). Comparison of the deduced amino acid sequencewith other amino acid sequences in GENBANK indicated that the proteinencoded by the ORF had strong homology (approximately 40%) to bothsubtilisin (Stahl et al., 1984, J. Bact., 158:411 ) and Isp-1 (Koide etal., 1986, J. Bact., 167:110) from B. subtilis 168. The most probableinitiation codon for this protease gene is the ATG at position 1 in FIG.6. This ATG (second codon in the ORF) is preceded by an excellentconsensus B. subtilis ribosome binding site (AAAGGAGATGA). In addition,the first 26 amino acids following this methionine resemble a typical B.subtilis signal sequence: a short sequence containing twopositively-charged amino acids, followed by 15 hydrophobic amino acids,a helix-breaking proline, and a typical Ala X Ala signal peptidasecleavage site (Perlman et al., 1983, J. Mol. Biol., 167:391).

Sequence analysis indicated that the ORF continued past the end of thedownstream EcoRV site, even though the 1.6 kb EcoRV fragment wassufficient to encode Epr protease activity. To map the 3' end of thegene, the DNA sequence of the overlapping KpnI to SalI fragment wasdetermined (FIG. 6). As shown in FIG. 2, the end of the ORF was found717 bp downstream of the EcoRV site and the entire epr gene was found toencode a 645 amino acid protein, the first approximately 380 amino acidsof which are homologous to subtilisin (FIG. 6). The C-terminalapproximately 240 amino acids are apparently not essential forproteolytic activity since N-terminal 405 amino acids encoded in the 1.6kb EcoRV fragment are sufficient for protease activity.

Structure of the epr Protein

In vitro transcription-translation experiments were used to confirm thesize of the protein. Plasmid pNP3 DNA (containing the 2.7 kb HaI-SalIfragment with the entire epr gene) was added to an S30-coupledtranscription/translation system (New England Nuclear) resulting in thesynthesis of a protein of approximately 75,000 daltons. (Additionalproteins of 60,000 and 34,000 daltons were also observed and presumablyrepresented processed or degraded forms of the 75,000 dalton protein.)This size agreed reasonably well with the predicted molecular weight of69,702 daltons for the primary product based on the deduced amino acidsequence.

The homology between the amino-terminal half of the epr protease andsubtilisin suggests that Epr might also be produced as a preproenzymewith a pro sequence of similar size to that of subtilisin (70-80 aminoacids). If true, and if there were no additional processing, this wouldargue that the mature Epr enzyme has a molecular weight of around58,000. Examination of culture supernatants, however, indicated that theprotein has a molecular weight of about 34,000. Comparison by SDS-PAGEof the proteins secreted by B. subtilis strain GP208 containing aplasmid with the epr gene (pNP3 or pNP5) or just the parent plasmidalone (pBs81/6) showed that the 2.7 kb HpaI-SalI fragment (FIG. 1)cloned in pNP3 directed the production of proteins of about 34,000 and38,000 daltons, whereas the 1.6 kb EcoRV fragment cloned in pNP5 in thesame orientation (FIG. 1) directed production of just the 34,000 daltonprotein. The two proteins appear to be different forms of the Eprprotease, resulting from either processing or proteolytic degradation.Clearly, the 1.6 kb EcoRV fragment, which lacks the 3' third of the eprgene, is capable of directing the production of an active proteasesimilar in size to that observed when the entire gene is present. Thissuggests that the protease normally undergoes C-terminal processing.

Bacillus strain GP208 containing the epr gene on plasmid pNP3 can beused to overproduce the Epr protease, which can then be purified byconventional procedures.

Location of epr on the B. subtilis Chromosome

To map epr on the B. subtilis chromosome, we introduced adrug-resistance marker into the chromosome at the site of the epr gene,and used phage PBS1-mediated transduction to determine the location ofthe insertion. A 1.3 kb EcoRI fragment containing a chloramphenicolacetyltransferase (cat) gene was cloned into the unique EcoRI site on anE. coli plasmid containing the epr gene (pNP2 is depicted in FIG. 7).The resulting plasmid (pNP7) was used to transform B. subtilis GP208,and chloramphenicol resistant transformants were selected. Since theplasmid cannot replicate autonomously in B. subtilis, the Cm^(r)transformants were expected to arise by virtue of a single, reciprocalrecombination event between the cloned epr gene on the plasmid and thechromosomal copy of the gene. Southern hybridization confirmed that thecat gene had integrated into the chromosome at the site of the clonedepr gene. Mapping experiments indicated that the inserted cat gene andepr gene are tightly linked to sacA321 (77% co-transduction), are weaklylinked to purA16 (5% co-transduction), and unlinked to hisA1. Thesefindings suggest that the epr gene is located near sacA in an area ofthe genetic map which does not contain any other known protease genes.

Construction of epr Deletion Mutant

To create a mutant Bacillus devoid of protease activity a deletion inthe 5' end of the cloned gene was constructed and then used to replacethe wild type gene in the chromosome. pNP2 was first digested withBamHI, which cleaves at a unique site within the epr gene, then thelinear plasmid DNA was treated with Bal31 exonuclease for 5 minutes at32° C., religated and transformed into E. coli JM107. Plasmid DNA wasisolated from 20 transformants, digested with EcoRI and HindIII toremove the epr gene insert and analyzed by gel electrophoresis. One ofthe plasmids had a 2.3 kb EcoRI-HindIII fragment replacing the 2.7 kbfragment indicating that approximately 400 base pairs had been deletedfrom the epr gene sequence. This plasmid was designated pNP8 (FIG. 7).This deletion mutant was introduced into B. subtilis GP208 by genereplacement methods as described above. The cat gene, contained on anEcoRI fragment from pEccI, was introduced into the EcoRI site on pNP8 tocreate pNP9 (FIG. 7). This E. coli plasmid was used to transform B.subtilis GP208 and Cm^(r) colonies were selected. Most of thetransformants produced a very small halo and the remaining 30% producedno halos on casein plates. The absence of a halo and therefore proteaseactivity resulted from a double crossover between chromosomal DNA andhomologous sequences from a concatemer of the plasmid DNA; these strainscontain the E. coli replicon and cat gene flanked by two copies of thedeleted epr gene. To screen for a strain that had undergone arecombination event between the two copies of the epr gene to resolvethe duplication, but which had jettisoned the cat gene and the E. colireplicon, a single colony was selected and grown overnight in richmedium without drug selection. Individual colonies arising from thisculture were then screened for drug resistance and about 0.1% of thesewere found to be Cm^(s). One such strain, GP216, containing deletionswithin the four protease genes (apr, npr, isp-1 and epr) was selectedfor further study.

The deletion in the chromosomal epr gene was confirmed by Southernhybridization. GP216, like the Cm^(r) parent strain, failed to produce ahalo on casein plates. In liquid cultures, however, ¹⁴ C-casein proteaseassays indicated that the epr mutation alone does not entirely eliminateresidual protease activity. A strain with deletions in epr, apr, npr,and isp, did not produce significantly less protease than a strain withmutations in just apr, npr, and isp. Finally, growth and sporulation ofthe quadruple protease deleted strain were assayed using standardlaboratory media. No differences were observed in growth in LB mediumwhen compared to the wild-type strain. Similarly, no appreciabledifferences were seen in sporulation frequency after growth on DSMmedium for 30 hours (1×10⁸ spores/ml for both GP208 and GP216).

Identification of Novel Proteolytic Activities

Strains of B. subtilis have been deleted for four non-essential proteasegenes, apr, npr, isp-1 and epr. These deletions reduce totalextracellular protease levels in culture supernatants of Spo+ hosts byabout 96% compared to the wild-type strain, but it is desirable todecrease or eliminate the remaining 4% residual protease activity forthe production of protease-labile products in Bacillus.

Using the azacoll assay, we have identified two novel proteases thataccount for this residual activity in GP227, a multiple proteasedeficient B. subtilis strain (aprΔ, nprΔ, eprΔ, isp-1Δ) which alsocontains a gene, sacQ*, encoding a regulatory protein. The sacQ* geneproduct functions by enhancing the production of degradative enzymes inBacillus, including the residual protease activity(s) and is the subjectof copending application U.S. Ser. No. 921,343, assigned to the sameassignee and hereby incorporated by reference. Due to enhancement bysacQ*, strain GP227 produces substantially more protease activity thanGP216, which lacks sacQ*.

In general, supernatants from cultures of B. subtilis GP227 wereconcentrated, fractionated by passage over a gel filtration column andassayed for protease activity. Two separate peaks of activity wereeluted from the column and designated RP-I and RP-II (residual protease)for the larger and smaller molecular weight species, respectively.Subsequent analysis of these two peaks confirmed that each accounted fora distinct enzymatic activity. The isolation and characterization of theRP-I and RP-II proteins, and the creation of a deletion mutation in eachof the RP-I and RP-II genes are described below.

Isolation and Characterization of RP-I

A simple and efficient purification scheme was developed for theisolation of RP-I from spent culture fluids. Cultures were grown inmodified MRS lactobacillus media (Difco, with maltose substituted forglucose) and concentrated approximately 10-fold using an Amicon CH2PRsystem equipped with a S1Y10 spiral cartridge. The concentratedsupernatant was dialyzed in place against 50 mM MES, 0.4M NaCl, pH 6.8,and fractionated over a SW3000 HPLC gel filtration column equilibratedwith the same buffer. The fractions containing protease activity wereidentified using a modification of the azocoll assay described above.

Fractions which were positive for the protease activity, correspondingto the higher molecular weight species, were pooled and concentratedusing a stirred cell equipped with a YM5 membrane, dialyzed vs. 50 mMMES, 100 mM KCl, pH 6.7 and applied to a benzamidine-Sepharose liquidaffinity column equilibrated with the same buffer. Most of the proteinapplied to the column (97%) failed to bind to the resin, however RP-Iprotein bound quantitatively and was eluted from the column with 250 mMKCl.

SDS-PAGE analysis of the benzamidine purified RP-I revealed that theprotein was greater than 95% homogeneous, and had a molecular weight ofapproximately 47,000 daltons. Purification by the above outlinedprocedure resulted in a 140-fold increase in specific activity, and anoverall recovery of about 10%.

Isoelectric focusing gels revealed that RP-I has a pI between 4.4 and4.7, indicating a high acidic/basic residue composition. The enzyme hasa pH optimum of 8.0 and a temperature maximum of 60° C. when azocoll isused as the substrate. It is completely inhibited by PMSF, indicatingthat it is a serine protease, but it is not inhibited by EDTA, even atconcentrations as high as 50 mM.

RP-I catalyzes the hydrolysis of protein substrates such as denaturedcollagen and casein, as well as ester substrates (0=C--O-- vs. O═C--N--linkages) such as N-α-benzolyl-L-arginine ethyl ester, phenylalaninemethylester, tyrosine ethyl ester and phenylalanine ethyl ester, butdoes not catalyze hydrolysis of the arginine peptide bond in thesynthetic substrate N-α-benzoyl-L-arginine-4-nitranilide. Collectively,these data demonstrate that RP-I is a serine endoproteinase that hasesterase activity and belongs to the subtilisin superfamily of serineproteases. Furthermore, these characteristics indicate that RP-I may bethe enzyme commonly referred to as Bacillopeptidase F (Boyer et al.,1968, Arch Biochem, Biophys., 128:442 and Roitsch et al., 1983, J.Bact., 155:145). Although Bacillopeptidase F has been reported to be aglycoprotein, we have not found carbohydrate to be associated with RP-I.

Cloning the Gene for RP-I

The sequence of the amino-terminal 28 amino acids of RP-I was determinedby sequential Edman degradation on an automatic gas phase sequenator andis depicted in FIG. 8. A DNA probe sequence (81 nucleotides) wassynthesized based on the most frequent codon usage for these amino acidsin B. subtilis (FIG. 8). The N-terminal amino acid sequence of RP-Icontains two tryptophan residues (positions 7 and 18). Since tryptophanhas no codon degeneracy, this facilitated the construction of a probethat was highly specific for the gene encoding RP-I.

High molecular weight DNA was isolated from B. subtilis strain GP216,digested with each of several different restriction endonucleases andfragments were separated by electrophoresis through a 0.8% agarose gel.The gel was blotted onto a nitrocellulose filter by the method ofSouthern (supra) and hybridized overnight with the ³² p end-labeledsynthetic RP-I specific probe under semi-stringent conditions (5X SSC,20% formamide, 1X Denhardts at 37° C.). Following hybridization, theblot was washed for one hour at room temperature in 2X SSC, 0.1% SDS.

The RP-I specific probe hybridized to only one band in each of therestriction digests indicating that the probe was specific for the RP-Igene. In the PstI digest, the probe hybridized to a 6.5 kb fragmentwhich was a convenient size for cloning and was also large enough tocontain most or all of the RP-I gene.

A clone bank containing PstI inserts in the 6-7 kb size range wasprepared from B. subtilis DNA as follows. Chromosomal DNA of strainGP216 was digested with PstI and separated on a 0.8% agarose gel. DNAfragments of 6-7 kb were purified from the gel by electroelution andligated with PstI digested pBR322 that had been treated with calfintestinal phosphatase to prevent recircularization of the vector upontreatment with ligase. The ligated DNA was transformed into competent E.coli DH5 cells and plated on media containing tetracycline.Approximately 3×10⁴ Tet^(r) transformants resulted, 80% of whichcontained plasmids with inserts in the 6-7 kb size range.

A set of 550 transformants was screened for the presence of the RP-Iinsert by colony hybridization with the ³² P-labeled RP-I specific probeand seven of these transformants were found to hybridize strongly withthe probe. Plasmid DNA was isolated from six of the positive clones andthe restriction digest patterns were analyzed with PstI and HindIII. Allsix clones had identical restriction patterns, and the plasmid from oneof them was designated pCR83.

Using a variety of restriction enzymes, the restriction map of pCR83insert shown in FIG. 9 was derived. The RP-I oligomer probe, whichencodes the N-terminal 28 amino acids of the mature RP-I protease, washybridized with restriction digests of pCR83 by the method of Southern(supra). The probe was found to hybridize with a 0.65 kb ClaI-EcoRVfragment suggesting that this fragment contained the 5' end of the gene.In order to determine the orientation of the RP-I gene, the strands ofthe ClaI-EcoRV fragment were separately cloned into the single-strandedphage M13. The M13 clones were then probed with the RP-I oligomer andthe results indicated that the RP-I gene is oriented in the leftward torightward direction according to the map in FIG. 9.

The DNA sequence of a portion of the PstI insert, as shown in FIG. 9,was determined, and an 81 base pair sequence (underlined in FIG. 10) wasfound that corresponded exactly with the sequence encoding the first 28amino acids of the protein. The BglII and ClaI sites designated in FIG.10 are identical to those designated in FIG. 9 and, in addition, theEcoRV site is identical to that designated in the restriction enzyme mapshown in FIG. 9. Portions of the untranslated region surrounding theRP-I coding region are also shown in FIG. 10; the DNA sequenceunderlined within the 5' untranslated region corresponds to the putativeribosome binding site.

The DNA sequence revealed an open reading frame that began atposition-15 (in FIG. 10) and proceeded through to position 2270. Themost probable initiation codon for this open reading frame is the ATG atposition 1 in FIG. 10. This ATG is preceded by a ribosome binding site(AAAGGGGGATGA), which had a calculated ΔG of -17.4 kcal. The first 29amino acids following this Met resemble a B. subtilis signal sequence,with a short sequence containing five positively-charged amino acids,followed by 16 hydrophobic residues, a helix-breaking proline, and atypical Ala-X-Ala signal peptidase cleavage site. After the likelysignal peptidase cleavage site, a "pro" region of 164 residues isfollowed by the beginning of the mature protein as confirmed by thedetermined N-terminal amino acid sequence. The first amino acid of theN-terminus, which was uncertain from the protein sequence, was confirmedas the Ala residue at position 583-585 from the DNA sequence. The entiremature protein was deduced to contain 496 amino acids with a predictedmolecular weight of 52,729 daltons. This size was in reasonableagreement with the determined molecular weight of the purified proteinof 47,000 daltons. In addition, the predicted isoelectric point of themature enzyme (4.04) was in good agreement with the observed pI of4.4-4.7. GENBANK revealed that the RP-I gene is partially homologous(30%) to subtilisin, to ISP-1 and, to a lesser extent (27%), to the eprgene product.

Cloning the RP-I Gene on a Multicopy Replicon

The PstI fragment was removed from pCR83 and ligated into PstIlinearized pBD9, a multicopy Bacillus replicon encoding erythromycin andkanamycin resistances. The ligated DNA was transformed into competentGP227 cells (the sacQ* enhancement strain) and kanamycin resistanttransformants were selected. A plasmid carrying the 6.5 kb PstI insertwas chosen and designated pCR88.

To confirm that this insert encoded the RP-I gene, GP227 cellscontaining pCR88 or pBD9 were grown in MRS medium under selectiveconditions for 50 hours at 37° C. Supernatant samples were collected andassayed for protease activity. Supernatants from the pCR88 culturescontained approximately 10-fold more protease activity than those fromthe pBD9 cultures. Furthermore, this secreted protease activity wasinhibited by PMSF and, when fractionated on a denaturing protein gel,the supernatant from the pCR88 sample contained an extra protein of 47kd. These results confirmed that the RP-I gene was encoded within the6.5 kb fragment, and that cloning the sequence in a multicopy repliconleads to the overproduction of the RP-I protein.

Location of the RP-I Gene on the B. Subtilis Chromosome

We mapped the location of the RP-I gene (bpr) on the B. subtilischromosome by integrating a drug resistance marker into the chromosomeat the site of bpr and using phage PBS1-mediated transduction todetermine the location of the cat insertion. A 1.3 kb SmaI fragmentcontaining a chloramphenicol acetyltransferase (cat) gene was clonedinto the unique EcoRV site of pCR92 (the 3.0 kb BglII of pCR83 clonedinto pUC18. The EcoRV site is in the coding region of bpr (FIG. 10). Theresulting plasmid, pAS112, was linearized by digestion with EcoR1 andthen used to transform B. subtilis strain GP216, andchloramphenicol-resistant transformants were selected (GP238). Cm^(r)transformants were expected to be the result of a double cross-overbetween the linear plasmid and the chromosome (marker replacement).Southern hybridization was used to confirm that the cat gene hadintegrated in the chromosome, interrupting the bpr gene. Mappingexperiments indicating that the inserted cat gene and bpr were stronglylinked to pyrD1 (89%) and weakly linked to metC (4%). The gene encodingthe neutral protease gene (npr) also maps in this region of thechromosome, although npr is less tightly linked to pyr (45% and 32%) andmore tightly linked to metC (18% and 21 %) than is bpr.

Construction of a Deleted Version of the RP-I Gene

An internal deletion in the RP-I sequence was generated in vitro.Deletion of the 650 bp sequence between the ClaI and EcoRV sites in thepCR83 insert removed the sequence encoding virtually the entireamino-terminal half of the mature RP-I protein. The deletion was made bythe following procedure.

The 4.5 kb PstI-EcoRI fragment of pCR78 (a pBR322 clone containing the6.5 kb PstI fragment) was isolated and ligated to pUC18 (a vectorcontaining the E. coli lacZ gene encoding B-galactosidase) that had beendigested with EcoRI and PstI. The ligation mix was then transformed intoE. coli DH5 cells. When plated onto LB media containing Xgal andampicillin, eight white colonies resulted, indicating insertion of thefragment within the gene encoding β-galactosidase. Plasmid DNA preparedfrom these colonies indicated that seven of the eight colonies containedplasmids with the 4.5 kb insert. One such plasmid, pKT2, was digestedwith EcoRV and ClaI, treated with Klenow fragment to blunt the ClaI endand then recircularized by self-ligation. The ligated DNA was thentransformed into E. coli DH5 cells. Approximately 100 transformantsresulted and plasmid DNA was isolated from Amp^(r) transformants andanalyzed by restriction digestion. Eight of eight clones had theClaI-EcoRV fragment deleted. One such plasmid was designated pKT2'. Thecat gene, carried on an EcoRI fragment from pEccI was then ligated intopKT2' for use in selecting Bacillus integrants as described above. Toinsert the cat gene, pKT2' was digested with EcoRI, treated with calfintestine alkaline phosphatase and ligated to a 1.3 kb EcoRI fragmentcontaining the cat gene. The ligated DNA was transformed into DH5 cellsand the Amp^(r) colonies that resulted were patched onto LB mediacontaining chloramphenicol. Two of 100 colonies were Cm⁵. Plasmid DNAwas isolated from these two clones and the presence of the 1.3 kb catgene fragment was confirmed by restriction enzyme analysis of plasmidDNA. One of these plasmids, pKT3, was used to introduce the deleted geneinto strain GP216 by gene replacement methods.

The DNA was transformed into GP216 and chloramphenicol resistantcolonies were selected. Chromosomal DNA was extracted from 8 Cm^(R)colonies and analyzed by Southern hybridization. One clone contained twocopies of the deleted RP-I gene resulting from a double crossoverbetween homologous sequences on the vector and in the chromosome. Theclone was grown in the absence of chloramphenicol selection and was thenreplica plated onto TBAB media containing chloramphenicol. One Cm^(s)colony was isolated and Southern analysis confirmed that the deletedgene had replaced the wild-type RP-I gene in the chromosome. This strainwas designated GP240. Analysis of supernatants from cultures of GP240confirmed the absence of RP-I activity.

Isolation and Characterization of RP-II

The purification scheme for RP-II was more extensive than for RP-Ibecause RP-II failed to bind benzamidine-Sepharose or otherprotease-affinity resins, e.g., arginine-Sepharose andhemoglobin-agarose, and we thus found it necessary to use moreconventional purification techniques such as ion exchangechromatography, gel filtration and polyacrylamide gel electrophoresis.

Concentrated crude supernatants of GP227 cultures were fractionated overDEAE-Sephacel (anion exchange) equilibrated at pH.6.8. At this pH theRP-II protein failed to bind the resin; however, approximately 80% ofthe total applied protein, including RP-I, bound the resin and was thusremoved from the sample. The column eluate was then fractionated bycation exchange chromatography using CM-Sepharose CL-6B equilibrated atpH 6.8. RP-II was capable of binding to the resin under these conditionsand was then eluted from the column with 0.5M KCl. To further enhancethe resolution of the cation exchange step, the RP-II eluate was thenrefractionated over a 4.6×250 mm WCX (weak cation exchange) HPLC columndeveloped with a linear gradient of NaCl. The WCX pool was thensize-fractionated over a TSK-125 HPLC column. The RP-II peak was thenfractionated a second time over the same column yielding a nearlyhomogeneous preparation of RP-II when analyzed by SDS-PAGE. The proteasewas purified over 6900-fold and represented approximately 0.01 % of thetotal protein in culture fluids of GP227. Alternatively, approximately30 fold more. RP-II can be purified from a Bacillus strain that is RP-Iand contains the sacQ* enhancing sequence (U.S. Ser. No. 921,343,assigned to the same assignee and hereby incorporated by reference),since the quantity of RP-II produced by such a strain is substantiallyincreased, representing about 0.3% of total protein in the culturefluid.

RP-II was insensitive to PMSF treatment, and therefore is not a serineprotease. SDS-PAGE analysis indicated that RP-II has a molecular mass of27.3 kd. The failure of RP-II to bind DEAE at pH 6.7 and PAE-300 (anHPLC anionic column) at pH 8.3 indicated that the protein has a basicisoelectric point which is greater than 8.3 (pI=8.7 bychromatofocusing). RP-II is highly sensitive to dithiothreitol (DTT, asulfhydryl reducing agent), being quantitatively inhibited at levels aslow as 1 mM in the azocoll assay. RP-II is also sensitive tocombinations of other sulfhydryl reagents with metal chelators (i.e.,mercaptoethanol with EDTA). Inhibition of proteases by sulfhydrylreagents is relatively rare and has only been described for a fewproteases, such as collagenase from C. histolyticum and carboxypeptidaseA. RP-II also possesses esterase activity as demonstrated by its abilityto hydrolyze phenylalanine methyl ester and n-t-BOC-L-glutamicacid-α-phenyl ester.

In order to obtain the cleanest possible sample of RP-II for sequenceanalysis, a final purification step was used which involved separationby polyacrylamide gel electrophoresis. Following electrophoresis,proteins were transferred electrophoretically from the gel to a sheet ofpolyvinylidene difluoride (PVDF) membrane. RP-II was visualized on thehydrophobic membrane as a "wet-spot" and the corresponding area was cutfrom the sheet and its amino-terminal amino acid sequence determined.

The sequence of the 15 amino acid terminal residues of RP-II(Ser-Ile-Ile-Gly-Thr-Asp-Glu-Arg-Thr-Arg-Ile-Ser-Ser-Thr-Thr-) is richin serine and arginine residues. Since both serine and arginine have ahigh degree of codon degeneracy, this increased the difficulty increating a highly specific probe. Therefore, additional amino acidsequence information was obtained from internal peptides that containedone or more non-degenerate amino acid residues.

Sequence Analysis of Internal Peptide Fragments of RP-II

Tryptic peptides from purified RP-II were produced and isolated usingreverse-phase HPLC. Since each of the amino acids tryptophan andmethionine is encoded by only one amino acid codon, a syntheticnucleotide probe, or "guess-mer" that encodes one or more of either ofthese amino acids will be highly specific for its complementarynucleotide sequences.

An HPLC chromatogram of the RP-II trypsin digested mixture was monitoredat three wavelengths: 210 nm (peptide bonds), 227 nm (aromatic residues,i.e., phenylalanine, tyrosine, tryptophan), and 292 nm (conjugated ringstructure of tryptophan). The 292 nm trace was used to identify peptidesof RP-II that contain a tryptophan residue. The 210 nm trace was used toobtain baseline resolved (i.e., single-species peptides) fragments forsequence analysis. Based on the 210 nm and 292 nm traces, threefragments were chosen for sequence analysis: T90, T94, and T92.Guess-mer oligomers were then synthesized based on the amino acidsequences of these fragments.

FIG. 11(a) is the amino-terminal sequence obtained for RP-II fragmentT90. A total of 15 residues were obtained, 67% of which have only one ortwo possible codons. The specificity of a probe (BRT90) constructedbased on the sequence of fragment T90 was enhanced by the presence of apredicted tryptophan residue (position 12). The number in parentheses ateach position represents the possible number of codons for each residue.

The amino-terminal sequence of RP-II fragment T94 is shown in FIG.11(b). Of the 30 residues determined, none were found to be tryptophan.Although only 36% of the residues (numbers 1-25) have two possiblecodons, the length of the corresponding 75-mer probe (707) renders ituseful for corroborating hybridization experiments conducted with theT90 probe.

The third and final probe was constructed based on sequence informationobtained from RP-II fragment T92 (FIG. 11(c)). Because of the relativelyhigh degree of degeneracy at the beginning and end of this sequence, aprobe was constructed based on residues 15-27. The resulting 39-merprobe (715) codes for a peptide of which half the residues have only oneor two possible codons. Furthermore, the specificity of this probe wasenhanced by the tandem location of a methionine and tryptophan residueat positions 26 and 27.

Cloning of RP-II

Chromosomal DNA was cut with various restriction enyzmes and a series ofhybridizations using the radiolabelled oligomer probes BRT90 and 707were performed. Both probes were labelled with ³² P and hybridized to aSouthern blot of GP241 DNA digested with BamHI, BglII, HincII, PstI, orEcoRI under semi-stringent conditions (5 x SSC, 10% formamide, 1 xDenhardt's, 100 μg/ml denatured salmon sperm DNA at 37° C.). Afterhybridization for 18 hours, the blots were washed with 2 x SSC, 0.1 %SDS for one hour at 37° C., and then washed with the same buffer at 45°C. for one hour. The results are shown in FIG. 12. Both probeshybridized to the same restriction fragments: HincII, ˜1 kb; PstI, 3-4kb, and EcoRI, 6-7 kb. The probes also hybridized to very largefragments in the BamHI and BglII-digested DNAs.

PstI fragments of 3-4 kb were used to construct a DNA library, asfollows. pBR322 was digested with PstI and treated with CIAP.Size-selected PstI-digested GP241 chromosomal DNA of 3-4.5 kb waselectroeluted from a 0.8% agarose gel. Approximately 0.1 μg of PstI-cutpBR322 and 0.2 μg of the size-selected DNA was ligated at 16° C.overnight. The ligated DNA was then transformed into E. coli DH5 cells.Approximately 10,000 colonies resulted, of which 60% contained plasmidswith the insert DNA. 1400 colonies were patched onto LB platescontaining 15 μg/ml tetracycline with nitrocellulose filters. Aftercolonies were grown at 37° C. overnight, the filters were processed tolyse the colonies, denature the DNA, and remove cell debris. The filterswere then baked at 80° for two hours. Colony hybridization was performedusing radiolabelled probe 707. Hybridization conditions were identicalto those used in the Southern blot experiments. Analysis of the plasmidDNA from four positive colonies identified one as containing plasmid DNAthat contained a 3.6 kb insert which strongly hybridized to both probes.The plasmid, pLP1, is shown in FIG. 13(b).

A restriction map of pLP1 (FIG. 13(a)) was constructed using a varietyof restriction endonucleases to digest pLP1, transferring thesize-fractionated digests onto nitrocellulose, and probing theimmobilized restriction fragments with the radiolabelled oligomersdescribed above. It was determined that all three oligomers, whichencode a total of 53 amino acids within the RP-II protein, hybridizedwith the 1.1 kb HincII fragment.

The 1.1 kb HincII fragment was isolated and cloned into M13mp18. A phageclone containing the HincII fragment was identified by hybridizationwith one of the oligomer probes. The DNA sequence of the HincII fragmentrevealed an open reading frame that spanned most of the fragment(position -24 to position 939 in. FIG. 14). The most probable initiationcodon for this open reading frame is the ATG at position 1 in FIG. 14.This ATG is preceded by a B. subtilis ribosome binding site (AAAGGAGG),which has a calculated ΔG of -16.0 kcal. The first 33 amino acidsfollowing this Met resembled a B. subtilis signal sequence, with a shortsequence containing four positively-charged amino acids, followed by 18hydrophobic residues, a helix-breaking proline, and a typical Ala-X-Alasignal peptidase cleavage site. After the presumed signal peptidasecleavage site, a "pro" region of 58 residues is found, followed by thebeginning of the mature protein as determined by the N-terminal aminoacid sequence of the purified protein. The amino terminal 16 residuesare underlined and designated "N-terminus". Amino acid sequences fromwhich the three guess-mers were deduced are also underlined anddesignated T94, T92, and T90. The determined amino acid sequences of thepeptides matched the deduced amino acid sequence except for a serineresidue encoded by nucleotides 379-381 and a cysteine residue encoded bynucleotides 391-393. The determined amino acid sequence predicted acysteine residue (position 14, T94 peptide) and an asparagine residue(position 18, T94 peptide), respectively (FIG. 11). The entire matureprotein was deduced to contain 221 amino acids with a predictedmolecular weight of 23,941 daltons. This size was in approximateagreement with the determined molecular weight of the purified protein28,000 daltons.

The deduced amino acid sequence showed only limited homology to othersequences in GENBANK. The strongest homology was to human protease E andbovine procarboxypeptidase A in a 25 amino acid sequence within RP-II(131-155, encoded by nucleotides 391-465; FIG. 14).

To further confirm the identity of the RP-II gene, the 3.6 kb PstIfragment was engineered onto a multi-copy Bacillus replicon to test foroverproduction of the RP-II protein. For this purpose the Bacillusplasmid pBs81/6 (Cm^(r), Neo^(r)) was inserted into the E. coli clonecontaining the RP-II gene. Plasmid pLP1 (8.0 kb) was digested withEcoRI, which cuts at a single site outside the PstI insert, and ligatedto EcoRI-digested pBs81/6 (4.5 kb; FIG. 13(a)). The resulting plasmid(pCR130) was used to transform GP241, and chloramphenicol orneomycin-resistant transformants were selected. Supernatant samples fromcultures of the transformants were found to contain 3-4 fold moreazocoll-hydrolyzing activity than the supernatants from cells containingonly the plasmid pBs81/6, indicating that the gene for RP-II is whollycontained within the 3.6 kb PstI fragment.

Location of the RP-II Gene on the B. subtilis Chromosome

In order to map the RPII gene (mpr) on the B. subtilis chromosome, weused B. subtilis strain GP261 described below which contained the catgene inserted into the chromosome at the site of the mpr gene and usedphage PBS1 transduction to determine the location of the cat insertion.

Mapping experiments indicated that the inserted cat gene and mpr werelinked to cysA14 (7% co-transduction) and to aroI906 (36%co-transduction) but unlinked to purA16 and da1. This data indicatedthat the mpr gene was between cysA and aroI in an area of the geneticmap not previously known to contain protease genes.

Deletion of the RP-II Gene on the Bacillus Chromosome

As described above for the other Bacillus subtilis proteases, an RP-IIBacillus deletion mutant was constructed by substituting a deletedversion of the RP-II gene for the complete copy on the chromosome. Toensure the deletion of the entire RP-II gene, a region of DNA wasdeleted between the two HpaI sites in the insert (FIG. 13(a)). Thisregion contains the entire 1.1 kb HincII fragment and an additional 0.9kb of DNA upstream of the HincII fragment.

To create the deletion, plasmid pLP1 (the pBR322 clone containing the3.6 kb PstI fragment) was digested with HpaI and size-fractionated on anagarose gel. Digestion of pLP1 results in the release of the 2 kbinternal HpaI fragment and a larger HpaI fragment containing the vectorbackbone and segments that flank the PstI insert (FIG. 13(c)). Thelarger HpaI fragment was purified and ligated with purified blunt-endedDNA fragments containing either the chloramphenicol-resistance (cat)gene from pMI1101 (Youngman et al., 1984, supra) or the bleomycinresistance (ble) gene from pKT4, a derivative of pUB110 (available fromthe Bacillus Stock Center, Columbus, Ohio).

The cat gene was isolated as a 1.6 kb SmaI fragment from pEcc1. This DNAwas ligated to the isolated large HpaI fragment of pLP1. The ligated DNAwas then transformed into E. coli DH5 cells. Approximately 20 Tet^(r)colonies resulted. One colony was found to be Cm^(r) when the colonieswere patched onto LB medium+5 μg/ml chloramphenicol. Analysis of theplasmid DNA from this colony confirmed the presence of the cat gene.This plasmid was called pLP2.

Plasmid pLP2 (FIG. 13(c)) was digested with PstI and then transformedinto GP241. This transformation gave approximately 280 Cm^(r) colonies;one colony was chosen for further study (GP261 ). Competent cells ofGP261 were prepared and then transformed with pDP104 (sacQ*); 10 Tet^(r)colonies resulted. Four colonies were grown in MRS medium and thepresence of sacQ* was confirmed by elevated levels of aminopeptidase.This strain was called GP262.

Since the cat gene was often used to select other vectors, a differentantibiotic resistance was also used to mark the deletion of the RP-IIgene on the Bacillus chromosome; i.e., the bleomycin-resistance gene ofpUB110. The ble gene was isolated from plasmid pKT4, a derivative ofpUB110, as an EcoRV-SmaI fragment and ligated to the purified large HpaIfragment (FIG. 13(c)) before tranformation into E. coli DH5 cells;tetracycline-resistant transformants were selected and then screened forresistance to phleomycin, a derivative of bleomycin, by patching ontoTBAB plates containing phleomycin at a final concentration of 2 μg/ml.Of 47 Tet^(r) transformants so screened, seven were alsophleomycin-resistant. The insertion of the ble gene was confirmed byrestriction analysis of the plasmids isolated from these clones. One ofthese plasmids, pCR125 (FIG. 13(c)), was used to introduce the deletedgene containing the ble gene marker into the strain GP241 by genereplacement methods, as described below.

Plasmid pCR125 was digested with EcoRI and the linear plasmid DNA wasused to transform GP241 to phleomycin resistance. Resistanttransformants were selected by plating the transformed cells onto TBABagar plates containing a gradient of 0-5 pg/ml phleomycin across theplate. Transformants that were resistant to approximately 2.5 μg/mlphleomycin on the plates were single-colony purified on TBAB phleomycinplates and thereafter grown on TBAB without selective antibiotic (strainGP263).

The strains bearing the RP-II deletion and the cat or ble insertion inthe RP-II gene, along with the positive regulatory element, sacQ*, wereevaluated for extracellular enzyme production, particularly protease andesterase activities.

The data given in Table 1, below, indicate that the presence of sacQ* inB. subtilis strain GP239, which bears null mutations in the fiveprotease genes apr (subtilisin), npr (neutral protease), epr(extracellular protease), isp (internal serine protease), and bpr,enhanced production of the RP-II protease (which also has esteraseactivity). To assess the influence on protease production of deletingRP-II from strains of B. subtilis bearing the sacQ* regulatory element,the following experiments were performed.

Independent clones of the RP-II deletion strain GP262 were shown toproduce negligible amounts of esterase activity and no detectable levelsof endoprotease activity using azocoll as substrate (Table I). Toconfirm the absence of protease activity, culture supernatants fromGP262 were concentrated to the extent that the equivalent of 1 ml ofsupernatant could be assayed. Even after 2.5 hours incubation of theequivalent of 1 ml of supernatant with the azocoll substrate, there wasno detectable protease activity in the deleted RP-II strain. Bycomparison, 50 μl of supernatant from GP239 typically gave an A₅₂₀ inthe azocoll assay of over 2.0 after a one hour incubation at 55° C. (Thepresence of sacQ* was confirmed by measurement of the levels ofaminopeptidase present in the culture fluids of this strain, which were50-80 fold higher than in analogous strains lacking sacQ*.) Thus,deletion of the two residual proteases, RP-I and RP-II, in Bacillusyields a strain that is largely incapable of producing extracellularendoproteases, as measured using azocoll as a substrate under theconditions described above.

                  TABLE 1    ______________________________________              Aminopeptidase  Protease                                      Esterase    Strain    (U/ml)          (U/ml)  (U/ml)    ______________________________________    GP238      0.04           0.13    0.02    GP239     1.7             84      1.16    GP262, AI 2.9             ND      0.08    GP262, AII              3.4             ND      0.11    GP262, BI 1.9             ND      0.10    GP262, BII              2.5             ND      0.10    ______________________________________

Aminopeptidase was measured using L-leucine-p-nitroanilide as substrate(1 unit=μmols substrate hydrolyzed/minute). Protease was measured usingthe standard azocoll assay (1 unit=ΔA₅₂₀ of 0.5/hour). Esterase wasmeasured using N-t-BOC-glutamic acid-α-phenyl ester as substrate (1 unit=μmols substrate hydrolyzed/minute). Strain GP238 has the genotype Δapr,Δnpr, Δepr, Δisp, Δrp-1; strain GP239 has the genotype Δapr, Δnpr, Δepr,Δisp, Δrp-1, sacQ*; and GP262 AI, AII, BI, and BII are independentclones of GP262 containing sacQ* and a cat insertional deletion inRP-II. ND means not detectable.

Referring to Table 2, several protease-deficient strains were alsotested for protease activity using the more sensitive resorufin-labelledcasein assay described earlier. As is shown in Table 2, although thestrain GP263, deleted for six protease genes, exhibited no detectableprotease activity in the azocoll test, such activity was detected in theresorufin-labelled casein test. GP271, the spoOA derivative of GP263,exhibited no detectable protease activity in either test, indicatingthat the prior protease activity detected in GP263 may be undersporulation control. The minor casein-detectable activity present inculture fluids of GP263 apparently belongs to the serine proteasefamily, because of its sensitivity to inhibition by PMSF. In thepresence of PMSF, no detectable protease activity was present incultures of GP263.

                  TABLE 2    ______________________________________                           Remaining                           activity                           (% of wild-type                           at t.sub.20)    Strain   Genotype            1      2    ______________________________________    1S75     Wild-type           100    100    GP202    .increment.apr, .increment.npr, amyE                                  5      8    GP208    .increment.apr, .increment.npr, .increment.isp-1, amyE,             met.sup.-            5      8    GP263    .increment.apr, .increment.npr, .increment.isp-1,             .increment.epr, .increment.bpr,                                 ND     0.5-1             .increment.mpr, .increment.hpr, amyE, met.sup.-    GP271    spoOA, .increment.apr, .increment.npr, .increment.isp-1,             .increment.epr,     ND     ND             .increment.bpr, .increment.mpr, .increment.hpr, amyE,    ______________________________________             met.sup.-     1 As measured using azocoll as substrate.     2 As measured using resorufin casein as substrate.

Other Embodiments

Other embodiments are within the following claims. For example, in someinstances it may be desirable to express, rather than mutate or delete,a gene or genes encoding protease(s) of the invention. This could bedone, for example, to produce the proteases for purposes such asimprovement of the cleaning activity of laundry detergents or for use inindustrial processes. This can be accomplished either by insertingregulatory DNA (any appropriate Bacillus promoter and, if desired,ribosome binding site and/or signal encoding sequence) upstream of theprotease-encoding gene or, alternatively, by inserting theprotease-encoding gene into a Bacillus expression or secretion vector;the vector can then be transformed into a Bacillus strain for production(or secretion) of the protease, which is then isolated by conventionaltechniques. Alternatively, the protease can be overproduced by insertingone or more copies of the protease gene on a vector into a host straincontaining a regulatory gene such as sacQ*.

We claim:
 1. An isolated and purified nucleic acid molecule encoding aBacillus residual protease II (mpr).
 2. A vector comprising a nucleicacid molecule encoding a Bacillus residual protease II (mpr).
 3. Thevector of claim 2, wherein said vector is an expression vector.
 4. Thevector of claim 2, wherein said vector is pLP1.
 5. The vector of claim 2wherein said vector is pCR130.
 6. A bacterial cell transformed with thevector of claim 2, 3, 4, or
 5. 7. A method for producing a Bacillusresidual protease II (mpr) comprising:(a) culturing the cell of claim 6under conditions which permit the expression of a said Bacillus residualprotease II, and (b) isolating said Bacillus residual protease II fromsaid cells cultured in step (a).